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Everyone knows that omega-3 polyunsaturated fatty acids (PUFAs) are good for you (eat your fish!), but no one really knows why. Although dietary PUFAs of both the omega-3 and omega-6 varieties are required for proper brain development, and may even stave off Alzheimer disease, little is known about their physiological targets. So it comes as welcome news that Frederic Darios and Bazbek Davletov at the MRC Laboratory of Molecular Biology (Cambridge, U.K.) have discovered a protein effector of PUFA action. In a paper in the April 6 Nature, the pair report that the omega-6 arachidonic acid and the omega-3 docosahexaenoic acid (DHA) stimulate neurite outgrowth in PC12 cells by binding to and activating syntaxin 3, a protein required for membrane expansion at the growth cone. Their results provide the first molecular explanation of how PUFAs regulate a defined event in neuronal cells, and suggest a way to screen for new compounds that stimulate neurite extension.

To investigate how the PUFA arachidonic acid (AA) promotes neurite outgrowth, Darios and Davletov used the familiar model of NGF-stimulated PC12 cells. In these cells, arachidonic acid is produced after NGF activates phospholipase A2, and the researchers showed that directly treating cells with phospholipase A2, or arachidonic acid itself, along with NGF, increased neurite extension. Lengthening of neurites involves membrane expansion by fusion of intracellular vesicles at the growth cone, which in turn requires SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor) proteins. So to pin down the role of fatty acids in the process, Darios and Davletov knocked down syntaxin-1, a SNARE protein that is sensitive to AA. Surprisingly, the RNAi knockdown had no effect on neurite outgrowth in response to NGF or AA. Instead, the authors found a related protein, syntaxin 3, was required for the AA response.

Syntaxins are known to partner with SNAPs (synaptosomal-associated proteins) during neurite outgrowth, and the researchers demonstrated that adding NGF or arachidonic acid to cells stimulated the association of syntaxin 3 with the SNAP25 protein. They replicated this interaction in a cell-free system with purified proteins and showed that adding arachidonic acid promoted formation of a stable syntaxin 3-SNAP25 complex. The concentration of AA required (100 μM) was consistent with the level they calculated to be present in growth cones upon phospholipase A2 activation. Protein structural analysis by circular dichroism (CD) indicated that arachidonic acid altered the conformation of syntaxin 3, but not SNAP25. The full SNARE complex that mediates membrane fusion contains an additional protein, synaptobrevin 2, and the researchers showed that formation of the ternary complex required AA activation of syntaxin 3.

In neurons, phospholipids commonly have PUFA side chains other than arachidonic acid. DHA, for instance, can account for as much as 8 percent of total brain lipid. Using their in vitro assay of protein association, the authors were able to test DHA and other fatty acids for their capacity to activate syntaxin 3. While saturated and mono-unsaturated fatty acids were ineffective, omega-6 (linoleic and arachidonic) and omega-3 (linolenic and DHA) PUFAs all stimulated syntaxin 3’s interaction with SNAP25 and induced the conformational change detected by CD. In vitro activity predicted neurite outgrowth: When tested on PC12 cells (at a single concentration of 200 μM), the two omega-3s stimulated neurite outgrowth to the same extent as arachidonic acid.

“The ability of syntaxin 3 to partner with other SNAREs strictly requires omega-3 and omega-6 PUFAs, which act as if to ‘oil’ the plasma membrane fusion machinery,” the authors write. They propose that PUFA binding to syntaxin 3 releases an inhibitory conformation and exposes a SNAP25 binding site. Because syntaxin 3 expression is ubiquitous, their findings may be applicable to membrane fusion in many kinds of cells, not just neurons.

Epidemiological evidence ties adequate intake of omega-3 PUFAs to a lower risk of Alzheimer disease, while experiments with AD mice show that DHA helps maintain synapses and reduce amyloid burden (see ARF related news story; Calon et al., 2005; and Lim et al., 2005). DHA also influences the activation of AKT, an important survival pathway for neurons (see ARF related news story). Whether or how activation of syntaxin relates to the reported beneficial effects of PUFAs in AD remains to be worked out. However, the ability of omega-3 and omega-6 PUFAs, but not other fatty acids, to activate syntaxin fits with the unique role of PUFAs in brain development and neuronal regeneration. The in vitro assay for syntaxin 3 activation will be useful for identifying new and potent PUFA mimics that might enhance neuronal growth.—Pat McCaffrey

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The recent paper of Frédéric Darios and Bazbek Davletov adds up to recent studies trying to decipher the molecular mechanism of action of neuroactive lipids such as omega-3 (n3) polyunsaturated fatty acids (PUFAs). Many cellular effectors of n3PUFAs have been previously identified (including neuroprotectin [1-2], cell signaling kinase Akt and PI3K [3-5], retinoid X receptors [6-7], etc.), but here the authors bring compelling in vitro evidence for a causal relationship linking PUFAs and syntaxin 3 to neurite growth. A key observation is the capacity of long-chain PUFAs (docosahexaenoic acid and arachidonic acid) to alter the 3D-structure of syntaxin 3 to make it complex with another synaptic protein, synaptosomal-associated protein-25. Among the series of fatty acids tested, only those altering syntaxin 3 conformation also promoted neurite growth. However, no distinction between n3 and n6 PUFAs was reported, contrasting with most in vivo studies, which favor n3PUFAs as neuroprotective agents. The next step is to determine if this scenario observed in cultured PC12 and hippocampal cells also occurs in vivo. If it does, it is possible that n3PUFAs exert their benefit on cognitive function through their action on syntaxin 3. This hypothesis will need to be tested in animal models of cognitive deficit. Syntaxin 3 could evolve as a potential drug target for Alzheimer disease.

We read this interesting paper, which offers a novel mechanism of action for PUFA-mediated neurite outgrowth. The mechanism involves fatty acid modulation of formation of syntaxin 3 complexes with SNARE proteins. The latter are required for regulating fusion of incoming vesicles with the expanding growth cone. Both the omega-6 and omega-3 fatty acids are similarly competent to promote syntaxin 3 protein complex formation and neurite outgrowth. Although high—100 micromolar half-maximal—concentrations of fatty acid are required for efficacy, the authors argue that these are achievable at the plasma membrane bilayer and they provide data for how the fatty acids impact physiological neurite outgrowth in living cells. Since Morris and collaborators have reported that low essential PUFA intake increases AD risk, these data suggest that one reason for this increased risk may be compromised neurite outgrowth and related loss of synapses. Increased oxidation of omega-3 and omega-6 fatty acids and their focal loss in AD is implied by measured increases in both F2 and F4 isoprostanes measured by multiple groups (Halliwell, Montine, Morrow, Pratico). However, these results showing similar impact from both omega-6 (AA) and omega-3 (DHA) cannot explain a selective benefit of omega-3 fatty acids or fish consumption as suggested by both epidemiology and AD animal model research, including work from our own lab, showing selective protection with DHA and adverse effects of high omega-6 (linoleic acid). Thus, more selective protection with DHA likely involves other mechanisms that may include reductions in amyloid accumulation, as seen in our data, as well as regulation of Akt translocation and activation as championed by Akbar et al., and generation of neuroprotectins like NPD1 as described by Nick Bazan's group. To our mind, it seems more and more probable that multiple protective mechanisms can be ascribed to these beneficial fats in your head.

The paper by Darios and Davletov provides significant information on the triggers of cell membrane expansion underlying neurite outgrowth. The importance of omega-3 and omega-6 polyunsaturated fatty acids in enabling syntaxin 3 to complex with SNAP25—eventually generating the SNARE ternary complex that results in membrane fusion and expansion—is a new and important finding. These results raise intriguing questions regarding the molecular events involved in AD pathogenesis. Recent research has shown that memory impairment in AD results from synaptic dysfunction with loss of LTP, in turn resulting from the toxic effects of Aβ oligomers at the cell membrane (Walsh et al., 2002; Cleary et al., 2005; Lesné et al., 2006). A recent paper by the group led by William Klein has proposed that such an effect is mediated by the specific interaction of Aβ oligomers with dendritic arbors at discrete puncta containing synaptic markers such as PSD-95, and possibly others including glutamate receptors, suggesting a clue to explain neuronal selectivity of damage (Lacor et al., 2004). Is it possible that Aβ aggregates may disrupt synaptic memory by interfering with synaptic expansion by the mechanisms outlined by Darios and Davletov? The possibility that Aβ oligomers interact with SNARE ternary complexes, or their precursors, and so compromise synaptogenesis is worth further investigation.

Another consideration concerns the effect of arachidonic acid and possibly other PUFAs in stabilizing a folding variant of syntaxin 3, causing it to interact with SNAP25. This is another example of a protein folding variant stabilized by a fatty acid. The HAMLET story has shown that a molecule of oleic acid interacts with α-lactalbumin, stabilizing a molten, globule-like folding variant of the latter, which is endowed with cytotoxic activity specific to tumor cells. A full understanding of how fatty acids, arising from specific metabolic modifications of membrane lipids, participate in membrane processing and/or membrane interactions with specific or nonspecific ligands is of fundamental importance to our understanding of the molecular basis of cell function and dysfunction.